In this paper, we prepared three types of transferrin-quantum dots conjugates (QDs-Tf) using three different methods (electrostatic interaction, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) coupling, denatured transferrin (dTf) coating). Fluorescence emission spectra, surface characteristics, zeta potentials of quantum dots (QDs) and QDs-Tf fluorescent probes were characterized by spectrophotometer, capillary electrophoresis, and dynamic light scattering. Fluorescent imaging of HeLa cells was also performed by QDs and QDs-Tf fluorescent probes. It was found that the fluorescence imaging performances of QDs-Tf probes prepared by electrostatic interaction and EDC coupling were better compared with the one prepared by dTf coating. Then a real-time single cell detection system was established to quantitatively evaluate cell labeling effects of QDs-Tf fluorescent probes. It was found that for cell labeling efficiency, the proportion of cells labeled by quantum dot probes to a group of cells, QDs-Tf probe prepared by EDC coupling showed the highest labeling efficiency (85.55±3.88%), followed by electrostatic interaction (78.86±9.57%), and dTf coating showed the lowest (40.09±10.2%). This efficiency order was confirmed by flow cytometry results. This study demonstrated the relationship between conjugation methods and the resultant QDs-Tf probes and provided a foundation for choosing appropriate QDs-Tf probes in cell labeling.